Exploring the effect of protein secondary structure on the solid state and physical stability of protein-based amorphous solid dispersions.

Amorphous solid dispersions (ASDs) using proteins as carriers have emerged as a promising strategy for stabilizing amorphous drug molecules. Proteins possess diverse three-dimensional structures that significantly influence their own properties and may also impact the properties of ASDs. We prepared ß-lactoglobulin (BLG) with different contents of ß-sheet and α-helical secondary structures by initially dissolving BLG in different mixed solvents, containing different ratios of water, methanol/ethanol, and acetic acid, followed by spray drying of the solutions. Our findings revealed that an increase in α-helical content resulted in a decrease in the glass transition temperature (Tg) of the protein. Subsequently, we utilized the corresponding mixed solvents to dissolve both BLG and the model drug celecoxib (CEL), allowing the preparation of ASDs containing either ß-sheet-rich or α-helix/random coil-rich BLG. Using spray drying, we successfully developed BLG-based ASDs with drug loadings ranging from 10 wt% to 90 wt%. At drug loadings below 40 wt%, samples prepared using both methods exhibited single-phase ASDs. However, heterogeneous systems formed when the drug loading exceeded 40 wt%. At higher drug loadings, physical stability assessments demonstrated that the α-helix/random coil-rich BLG structure exerted a more pronounced stabilizing effect on the drug-rich phase compared to the ß-sheet-rich BLG. Overall, our results highlight the importance of considering protein secondary structure in the design of ASDs.

Investigating the influence of protein secondary structure on the dissolution behavior of ß-lactoglobulin-based amorphous solid dispersions.

Proteins acting as carriers in amorphous solid dispersions (ASDs) demonstrate a notable sensitivity to the spray drying process, potentially leading to changes in their conformation. The main aim of this study was to investigate the dissolution performance of ASDs based on proteins with different content of secondary structures, specifically ß-sheet and α-helix structures. We prepared ß-sheet-rich and α-helix-rich ß-lactoglobulin (BLG), along with corresponding ASDs containing 10 wt% and 30 wt% drug loadings, through spray drying using celecoxib as the model drug. Circular dichroism and Fourier Transform Infrared Spectroscopy results revealed that even though changes in secondary structure were obtained in the spray-dried powders, the BLGs exhibited reversibility upon re-dissolving in phosphate buffer with varying pH levels. Both ß-sheet-rich BLG and α-helix-rich BLG exhibited enhanced dissolution rates and higher solubility in the media with pH values far from the isoelectric point (pI) of BLG (pH 2, 7, 8, and 9) compared to the pH closer to the pI (pH 3, 4, 5, and 6). Notably, the release rate and solubility of the drug and BLG from both types of BLG-based ASDs at 10 wt% drug loading were largely dependent on the solubility of pure SD-BLGs. α-helix-rich BLG-ASDs consistently exhibited equivalent or superior performance to ß-sheet-rich BLG-ASDs in terms of drug release rate and solubility, regardless of drug loading. Moreover, both types of BLG-based ASDs at 10 wt% drug loading exhibited faster release rates and higher solubility, for both the drug and BLG, compared to the ASDs at 30 wt% drug loading in pHs 2, 7, and 9 media.

Mechanisms of Drug Solubility Enhancement Induced by ß-Lactoglobulin-Based Amorphous Solid Dispersions.

Protein-based amorphous solid dispersions (ASDs) have emerged as a promising approach for enhancing solubility in comparison to crystalline drugs. The dissolution behavior of protein-based amorphous solid dispersions (ASDs) was investigated in various pH media. ASDs of four poorly soluble model drugs with acidic (furosemide and indomethacin), basic (carvedilol), and neutral (celecoxib) properties were prepared by spray drying at 30 wt % drug loading with the protein ß-lactoglobulin (BLG). The effect of spray-dried BLG (SD-BLG) solubility and protein binding ability with dissolved drugs in solution were investigated to retrieve the mechanisms governing the improvement of drug solubility from the BLG-based ASDs. Powder dissolution results showed that all ASDs obtained a higher maximum concentration (Cmax) compared to the respective pure crystalline drugs. It was found that the solubility increase of the drugs from the ASDs was to a large extent dependent on the solubility of the pure SD-BLG at the investigated pH values (low solubility at pH near the isoelectric point (pI) of BLG). Furthermore, drug-protein interactions in a solution were observed, in particular at pH values where the drugs were neutral. These drug-protein interactions also resulted, to some extent, in the stabilization of the drug in supersaturation.

Protein Based Amorphous Solid Dispersion: a Case Study Investigating Different Whey Proteins at High Drug Loading.

PURPOSE: Whey protein isolate (WPI) has previously been shown to be a promising new excipient for the development of amorphous solid dispersions (ASD) at a high drug loading of 50% (w/w). Whilst WPI is a protein mixture, comprising mainly the three proteins ß-lactoglobulin (BLG), α-lactalbumin (ALA), casein glycomacropeptides (CGMP), the individual contributions of these three proteins to the overall performance of whey protein based ASDs has still not been investigated. In addition, the limitations of the technology at even higher drug loadings (i.e., more than 50%) have not yet been explored. In this study, BLG, ALA, CGMP and WPI were each prepared as ASDs with the two poorly water-soluble drugs (Compound A and Compound B) at 50%, 60% and 70% drug loadings. METHODS: Solid state characterization, dissolution rate and physical stability of the obtained samples were analyzed. RESULTS: All the obtained samples were amorphous and showed faster dissolution rates compared to the respective pure crystalline drugs. However, the BLG based formulations-at least for Compound A-were outperforming the other ASDs in terms of stability, dissolution enhancement and solubility increase. CONCLUSION: Overall, the study confirmed that the investigated whey proteins showed their potential in developing ASDs even at high drug loadings of up to 70%.

Enhanced dissolution rate of nimodipine through ß-lactoglobulin based formulation.

Amorphous solid dispersions (ASD) have been considered as one of the most effective strategies to increase solubility and dissolution rate of poorly water-soluble drugs. Carriers, in which the poorly water-soluble drug is dispersed, contribute a large extent to the solid-state properties, stabilities and dissolution performance of ASDs. This study investigated the solid-state properties, physical stability, and in vitro dissolution behaviour of nimodipine ASDs formulated with a traditional polymeric carrier, i.e., polyvinylpyrrolidone (PVP) and a novel carrier, i.e., ß-lactoglobulin (BLG). The ASDs with both carriers were prepared using ball milling as preparative technique at 10 %, 17.5 %, 25 %, 30 % and 40 % drug loadings (DLs). All the formulations were found to be amorphous upon milling for 60 min based on X-ray powder diffraction measurements, however, the ASDs were found to be homogeneous unequivocally only at DLs below 25 %. After open storage at accelerated conditions (40 °C/75 % relative humidity), only the ASDs formulated with BLG at 10 % and 17.5 % DLs maintained the amorphous form. The dissolution study revealed that all the freshly prepared ASDs formulated with PVP and the ASDs formulated with BLG at or above 25 % DLs, showed a low drug release (<30 µg/mL in simulated gastric fluid, < 70 µg/mL in simulated intestinal fluid). Whilst the ASD formulated with BLG at 10 % DL exhibited a high drug release with a maximum concentration (Cmax) of 251 µg/mL in simulated gastric fluid and 231 µg/mL in simulated intestinal fluid. Surprisingly, the ASD formulated with BLG at 17.5 % DL demonstrated an even higher drug release (Cmax, 643 µg/mL in simulated gastric fluid, 332 µg/mL in simulated intestinal fluid), compared to the ASD of 10 % DL. These findings underline the importance of rationally investigating both carrier types and DL in the design of ASDs, in order to obtain a stable ASD with the desired enhanced dissolution rate of poorly water-soluble drugs.

Stabilizing Mechanisms of ß-Lactoglobulin in Amorphous Solid Dispersions of Indomethacin.

Proteins, and in particular whey proteins, have recently been introduced as a promising excipient class for stabilizing amorphous solid dispersions. However, despite the efficacy of the approach, the molecular mechanisms behind the stabilization of the drug in the amorphous form are not yet understood. To investigate these, we used experimental and computational techniques to study the impact of drug loading on the stability of protein-stabilized amorphous formulations. ß-Lactoglobulin, a major component of whey, was chosen as a model protein and indomethacin as a model drug. Samples, prepared by either ball milling or spray drying, formed single-phase amorphous solid dispersions with one glass transition temperature at drug loadings lower than 40-50%; however, a second glass transition temperature appeared at drug loadings higher than 40-50%. Using molecular dynamics simulations, we found that a drug-rich phase occurred at a loading of 40-50% and higher, in agreement with the experimental data. The simulations revealed that the mechanisms of the indomethacin stabilization by ß-lactoglobulin were a combination of (a) reduced mobility of the drug molecules in the first drug shell and (b) hydrogen-bond networks. These networks, formed mostly by glutamic and aspartic acids, are situated at the ß-lactoglobulin surface, and dependent on the drug loading (>40%), propagated into the second and subsequent drug layers. The simulations indicate that the reduced mobility dominates at low (<40%) drug loadings, whereas hydrogen-bond networks dominate at loadings up to 75%. The computer simulation results agreed with the experimental physical stability data, which showed a significant stabilization effect up to a drug fraction of 70% under dry storage. However, under humid conditions, stabilization was only sufficient for drug loadings up to 50%, confirming the detrimental effect of humidity on the stability of protein-stabilized amorphous formulations.

A Comparative Study between A Protein Based Amorphous Formulation and Other Dissolution Rate Enhancing Approaches: A Case Study with Rifaximin.

Amorphous solid dispersions (ASDs) based on proteins as co-formers have previously shown promising potential to improve the solubility and bioavailability of poorly water-soluble drugs. In particular, whey proteins have shown to be promising co-formers and amorphous stabilizers in ASD formulations, including at high drug loading. In this study, the feasibility of the whey protein ß-lactoglobulin (BLG) as a co-former in ASDs was compared to the more traditional ASD co-formers based on synthetic polymers (hydroxypropyl methylcellulose acetate succinate and Eudragit® L) as well as to a nanocrystalline formulation. The poorly water-soluble drug rifaximin (RFX) was chosen as the model drug. All drug/co-former formulations were prepared as fully amorphous ASDs by spray drying at 50% (w/w) drug loading. The BLG-based ASD had the highest glass transition temperature and showed a faster dissolution rate and higher drug solubility in three release media with different pH values (1.2, 4.5, and 6.5) compared to the polymer-based ASDs and the nanocrystalline RFX. In conclusion, BLG is a promising co-former and amorphous stabilizer of RFX in ASD formulations, superior to the selected polymer-based ASD systems or the nanocrystalline formulation.

Design of Inhalable Solid Dosage Forms of Budesonide and Theophylline for Pulmonary Combination Therapy.

Corticosteroid resistance poses a major challenge to effective treatment of chronic obstructive pulmonary diseases. However, corticosteroid resistance can be overcome by co-administration of theophylline. The aim of this study was to formulate the corticosteroid budesonide with theophylline into inhalable dry powders intended for pulmonary combination therapy. Four types of spray-dried powders were prepared: (i) budesonide and theophylline co-dissolved and processed using a 2-fluid nozzle spray drier, (ii) budesonide nanocrystals and dissolved theophylline co-dispersed and processed using a 2-fluid nozzle spray drier, (iii) dissolved budesonide and dissolved theophylline processed using a 3-fluid nozzle spray drier, and (iv) budesonide nanocrystals and dissolved theophylline processed using a 3-fluid nozzle spray drier. Spray drying from the solutions resulted in co-amorphous (i) and partially amorphous powders (iii), whereas spray drying of the nanosuspensions resulted in crystalline products (ii and iv). Even though budesonide was amorphous in (i) and (iii), it failed to exhibit any dissolution advantage over the unprocessed budesonide. In contrast, the dissolution of budesonide from its nanocrystalline formulations, i.e., (ii) and (iv), was significantly higher compared to a physical mixture or unprocessed budesonide. Furthermore, the spray-dried powders obtained from the 2-fluid nozzle spray drier, i.e., (i) and (ii), exhibited co-deposition of budesonide and theophylline at the same weight ratio in the aerodynamic assessment using the New Generation Impactor. In contrast, the depositions of budesonide and theophylline deviated from the starting weight ratio in the aerodynamic assessment of spray-dried powders obtained from the 3-fluid nozzle spray drier, i.e., (iii) and (iv). Based on these results, the powders spray-dried from the suspension by using the 2-fluid nozzle spray drier, i.e., (ii), offered the best formulation properties given the physically stable crystalline solid-state properties and the co-deposition profile.

Formulating Inhalable Dry Powders Using Two-Fluid and Three-Fluid Nozzle Spray Drying.

PURPOSE: The spray drying process is widely applied for pharmaceutical particle engineering. The purpose of this study was to investigate advantages and disadvantages of two-fluid nozzle and three-fluid nozzle spray drying processes to formulate inhalable dry powders. METHODS: Budesonide nanocomposite microparticles (BNMs) were prepared by co-spray drying of budesonide nanocrystals suspended in an aqueous mannitol solution by using a two-fluid nozzle spray drying process. Budesonide-mannitol microparticles (BMMs) were prepared by concomitant spray drying of a budesonide solution and an aqueous mannitol solution using a spray drier equipped with a three-fluid nozzle. The resulting dry powders were characterized by using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA) and Raman microscopy. A Next Generation Impactor was used to evaluate the aerodynamic performance of the dry powders. RESULTS: XRPD and DMA results showed that budesonide remained crystalline in the BNMs, whereas budesonide was amorphous in the BMMs. Spray drying of mannitol into microparticles resulted in a crystalline transformation of mannitol, evident from XRPD, DSC and Raman spectroscopy analyses. Both BMMs and BNMs displayed a faster dissolution rate than bulk budesonide. The yield of BNMs was higher than that of BMMs. The mass ratio between budesonide and mannitol was preserved in the BNMs, whereas the mass ratio in the BMMs was higher than the theoretical ratio. CONCLUSIONS: Spray drying is an enabling technique for preparation of budesonide amorphous solid dispersions and nanocrystal-embedded microparticles. Two-fluid nozzle spray drying is superior to three-fluid nozzle spray drying in terms of yield.

Mixed micellar system stabilized with saponins for oral delivery of vitamin K.

Poorly soluble vitamin K cannot be absorbed by patients suffering from cholestasis due to extremely low level of bile salts in the intestine. A formulation of vitamin K including glycocholic acid (i.e. Konakion® MM), does not increase bioavailability because it is unstable due to protonation of glycocholic acid at gastric pH. To develop a stable formulation, saponins were introduced as neutral surfactants to (partly) replace glycocholic acid. Experimental design was made to investigate the effect of the composition on particle size at neutral pH and upon acidification at pH 1.5. Two formulations that were within the optimized composition window were loaded with vitamin K and those showed superior stability at low pH as compared to Konakion® MM: sizes were between 43 and 46 nm at pH 7.3 and between 46 and 58 nm after 1 h incubation at pH 1.5, respectively, but large aggregates were formed at pH 1.5 in presence of Konakion® MM. Micelles were cytocompatible with Caco-2 cells at concentration of surfactants (saponins and glycocholic acid) up to 0.15 mg/ml. Uptake of vitamin K by Caco-2 cells was 4.2-4.9 nmol/mg protein for saponins-containing formulations and 7.1 nmol/mg protein for Konakion® MM. This, together with the superior stability at low pH, makes saponins-containing mixed micelles promising oral formulations for vitamin K.

Engineering of budesonide-loaded lipid-polymer hybrid nanoparticles using a quality-by-design approach.

Chronic obstructive pulmonary disease (COPD) is a complex disease, characterized by persistent airflow limitation and chronic inflammation. The purpose of this study was to design lipid-polymer hybrid nanoparticles (LPNs) loaded with the corticosteroid, budesonide, which could potentially be combined with small interfering RNA (siRNA) for COPD management. Here, we prepared LPNs based on the biodegradable polymer poly(dl-lactic-co-glycolic acid) (PLGA) and the cationic lipid dioleyltrimethylammonium propane (DOTAP) using a double emulsion solvent evaporation method. A quality-by-design (QbD) approach was adopted to define the optimal formulation parameters. The quality target product profile (QTPP) of the LPNs was identified based on risk assessment. Two critical formulation parameters (CFPs) were identified, including the theoretical budesonide loading and the theoretical DOTAP loading. The CFPs were linked to critical quality attributes (CQAs), which included the intensity-based hydrodynamic particle diameter (z-average), the polydispersity index (PDI), the zeta-potential, the budesonide encapsulation efficiency, the actual budesonide loading and the DOTAP encapsulation efficiency. A response surface methodology (RSM) was applied for the experimental design to evaluate the influence of the CFPs on the CQAs, and to identify the optimal operation space (OOS). All nanoparticle dispersions displayed monodisperse size distributions (PDI<0.2) with z-averages of approximately 150nm, suggesting that the size is not dependent on the investigated CFPs. In contrast, the zeta-potential was highly dependent on the theoretical DOTAP loading. Upon increased DOTAP loading, the zeta-potential reached a maximal point, after which it remained stable at the maximum value. This suggests that the LPN surface is covered by DOTAP, and that the DOTAP loading is saturable. The actual budesonide loading of the LPNs was mainly dependent on the initial amount of budesonide, and a clear positive effect was observed, which shows that the interaction between drug and PLGA increases when increasing the initial amount of budesonide. The OOS was modeled by applying the QTPP. The OOS had a budesonide encapsulation efficiency higher than 30%, a budesonide loading above 15µg budesonide/mg PLGA, a zeta-potential higher than 35mV and a DOTAP encapsulation efficiency above 50%. This study shows the importance of systematic formulation design for understanding the effect of formulation parameters on the characteristics of LPNs, eventually resulting in the identification of an OOS.

Advances in combination therapy of lung cancer: Rationales, delivery technologies and dosage regimens.

Lung cancer is a complex disease caused by a multitude of genetic and environmental factors. The progression of lung cancer involves dynamic changes in the genome and a complex network of interactions between cancer cells with multiple, distinct cell types that form tumors. Combination therapy using different pharmaceuticals has been proven highly effective due to the ability to affect multiple cellular pathways involved in the disease progression. However, the currently used drug combination designs are primarily based on empirical clinical studies, and little attention has been given to dosage regimens, i.e. how administration routes, onsets, and durations of the combinations influence the therapeutic outcome. This is partly because combination therapy is challenged by distinct physicochemical properties and in vivo pharmacokinetics/pharmacodynamics of the individual pharmaceuticals, including small molecule drugs and biopharmaceuticals, which make the optimization of dosing and administration schedule challenging. This article reviews the recent advances in the design and development of combinations of pharmaceuticals for the treatment of lung cancer. Focus is primarily on rationales for the selection of specific combination therapies for lung cancer treatment, and state of the art of delivery technologies and dosage regimens for the combinations, tested in preclinical and clinical trials.

Development and comparison of intramuscularly long-acting paliperidone palmitate nanosuspensions with different particle size.

The main purpose of this study was to develop and compare the pharmacokinetic behavior of two paliperidone palmitate (PP) nanosuspensions with different particle size after intramuscular (i.m.) administration. PP nanosuspensions were prepared by wet media milling method and the mean particle size of nanosuspension was controlled as 1,041 ± 6 nm (A) and 505 ± 9 nm (B), respectively. The morphology of nanosuspensions was observed by scanning electron microscope (SEM). Differential scanning calorimeter (DSC) and powder X-ray diffraction (PXRD) confirmed the crystallinity of PP in nanosuspensions. The physical and chemical stabilities of nanosuspensions A and B were investigated by particle analyzer and HPLC after storage for 2 months at 25°C, 4°C and mechanical shaking condition. No obvious change in particle size and chemical degradation of drug were observed. Following single-dose i.m. administration to beagle dogs, the release of paliperidone lasted for nearly 1 month. The Tmax of nanosuspensions A and B was 6 (d) and 10 (d). The AUC0-t and Cmax of nanosuspensions A was 2.0-fold and 1.8-fold higher than nanosuspensions B (p<0.05). The results demonstrated that PP nanosuspensions formulation had long-acting effect. Nanosuspension A with a larger particle size performed better than nanosuspension B. As a result, it is important to design appropriate particle size of nanosuspensions for i.m. administration in order to produce larger therapeutic effect.

Improved oral bioavailability of glyburide by a self-nanoemulsifying drug delivery system.

AIM: The present study aimed at the development and characterisation of self-nanoemulsifying drug delivery system (SNEDDS) to improve the oral bioavailability of poorly soluble glyburide. METHODS: The solubility of glyburide was determined in various oils, surfactants and co-surfactants which were grouped into two different combinations to construct ternary phase diagrams. The formulations were evaluated for emulsification time, droplet size, zeta-potential, electrical conductivity and stability of nanoemulsions. RESULT: The optimised SNEDDS loading with 5 mg/g glyburide comprised 55% Cremophor® RH 40, 15% propanediol and 30% Miglyol® 812, which rapidly formed fine oil-in-water nanoemulsions with 46 ± 4 nm particle size. Compared with the commercial micronised tablets (Glynase®PresTab®), enhanced in vitro release profiles of SNEDDS were observed, resulting in the 1.5-fold increase of AUC following oral administration of SNEDDS in fasting beagle dogs. CONCLUSIONS: These results indicated that SNEDDS is a promising drug delivery system for increasing the oral bioavailability of glyburide.

Star-shape copolymer of lysine-linked di-tocopherol polyethylene glycol 2000 succinate for doxorubicin delivery with reversal of multidrug resistance.

A star-shape copolymer of nanostructure-forming material, P-glycoprotein (P-gp) reversible inhibitor and anticancer enhancer, lysine-linked di-tocopherol polyethylene glycol 2000 succinate (PLV(2K)), was synthesized to overcome multidrug resistance (MDR) in cancer chemotherapy. The critical micellar concentration of PLV(2K) was as low as 1.14 µg/mL, which can endow nanoassemblies good physical stability. Doxorubicin (DOX) was encapsulated into the hydrophobic core of PLV(2K) (PLV(2K)-DOX), with encapsulation efficiency as high as 94.5% and a particle size of 16.4 nm. DOX released from PLV(2K)-DOX nanomicelles was pH-dependent, which ensures micelles stable in blood circulation and releases DOX within tumor cells. Facilitated by the cytotoxicity and uncompetitive P-gp ATPase inhibition by PLV(2K), PLV(2K)-DOX showed greater cytotoxicity compared with DOX solution with increased intracellular accumulation in resistant MCF-7/Adr cells. PLV(2K)-DOX nanomicelles were uptaken into MCF-7/Adr cells via macropinocytosis and caveolae-mediated endocytosis, which further facilitate escapement of P-gp efflux. The anticancer efficacy in vivo was evaluated in 4T1-bearing mice and inhibition of tumor by PLV(2K)-DOX was more effective than TPGS-DOX and DOX solution. In summary, PLV(2K) copolymer has striking functions such as uncompetitive P-gp ATPase reversible inhibitor and anticancer efficacy, and could be a promising nanocarrier in improving the chemotherapy of hydrophobic anticancer drugs.